The Id proteins are a family of four related proteins implicated in the control of differentiation and cell cycle progression in organisms ranging from flies to man [Ruzinova et al., Trends Cell Biol; 13:410-418, (2003), Hasskarl et al., Cancer Biol Ther; 1:91-6, (2002), Lasorella et al., Oncogene; 20:8326-33 (2001), Sikder et al., Cancer Cell; 3:525-30 (2003), Fong et al., Trends Mol Med; 10: 387-92 (2004) and Zebedee and Hara, Oncogene; 20: 8317-25(2001)]. The four Id proteins, Id1, Id2, Id3 and Id4, affect control by sequestering basic helix-loop-helix (bHLH) transcription factors and forming heterodimers that are unable to bind to DNA [Benezra et al., Cell, 61:49-59 (1990), O'Toole et al., J Biol Chem; 278: 45770-62003 (2003), Lister and Baron, Gene Expr; 7: 25-38 (1998) and Yates et al., Embo J; 18:968-76 (1999)]. In this regard, biochemical and genetic data have established that the target of the Id proteins are the ubiquitously expressed bHLH proteins referred to as E proteins such as E12, E47, E2-2 and HEB as well as certain tissue-restricted HLH proteins such as MyoD, MRFs, etc., that control cell type specific gene expression and the expression of cell cycle regulatory genes [Lassar et al., Curr Opin Cell Biol; 6:788-94 (1994) and Langlands et al., J Biol Chem; 272: 19785-93 (1997)]. The structural mediator of this sequestering process is a highly conserved dimerization motif known as the helix-loop-helix (HLH) domain that is common to all four proteins [Riechmann et al., Nucleic Acids Res; 22:749-55 (1994) and Langlands et al., J Biol Chem; 272: 19785-93 (1997)]. The bHLH proteins contain a cluster of amino acids rich in basic residues adjacent to the HLH dimerization motif which mediates DNA binding of homodimeric or heterodimeric HLH complexes. Since the Id proteins lack a basic DNA binding domain, the heterodimers between Id and bHLH proteins cannot bind DNA. This dominant negative mode of inhibition of DNA binding activity is widely used in the cell and is also employed by members of the leucine zipper and homeodomain protein families [Ron and Habener, Genes Dev; 6: 439-453 (1992)].
Id1 and Id3 are co-expressed temporally and spatially during mouse neurogenesis and in tissues undergoing active morphogenesis [Duncan et al., Dev. Biol; 154:1-10 (1992), Ellmeier, et al., Dev Dyn; 203:163-73 (1995) and Jen, Y., et al., Dev Dyn; 208:92-106 (1997)]. Id1 and Id3 are also important to neovascularization, a process important to the growth, progression and metastasis of tumors [Dhanabal et al., Curr. Med. Chem. Anticancer Agents; 5: 115-30 (2005) and Ferrara et al., Nat Rev Drug Discov; 3: 391-400 (2004)]. The loss of the Id1 and Id3 genes (knockout mouse) results in impaired neovascularization and diminished ability to support tumor growth in animals [Lyden et al., Nature, 401:670-7 (1999) and Lyden et al., Nat Med; 7: 1194-201 (2001)]. Consistent with this observed anti-tumor activity, the regulation of many biochemicals important to angiogenesis like MMP2 and certain integrins are influenced by Id proteins [Lyden et al., Nature, 401:670-7 (1999) and Ruzinova et al., Cancer Cell; 4: 277-89 (2003)].
Breast cancer is one of many pathologies illustrating the important role of Id genes and proteins in promoting cell proliferation and negatively regulating differentiation [de Candia et al., Adv Cancer Res; 92: 81-94 (2004), Coletta et al., J Mammary Gland Biol Neoplasia; 9: 39-53 (2004), Desprez et al., J Mammary Gland Biol Neoplasia; 8: 225-39 (2003) and Fong et al., Proc Natl Acad Sci USA; 100: 13543-8 (2003)]. High levels of Id gene expression have also been observed in tumor cell lines derived from different tissues. In accordance with this, one of the members of this gene family, Id1, has been shown to promote proliferation and inhibit functional differentiation of mouse mammary epithelial cells (SCp2 cells), maintained in cell culture [Desprez et al., Mol Cell Biol, 15:3398-3404 (1995)]. In addition, Id1 deletion totally blocks tumor formation in an animal model of human breast cancer when combined with treatment with an HSP-90 inhibitor [de Candia P et al., Proc Natl Acad Sci USA; 100: 12337-42 (2003)]. Experiments establishing the role of particular Id proteins in a specific type of cancer are heavily dependent on analysis by immunohistochemistry (IHC) of specific tumor samples. For example, normal mammary gland is composed of several cell types, but it is the luminal epithelial cells, which line the inside of ducts and the lobules, that are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of any regulatory factor in mammary biology and its significance to carcinogenesis, it is essential to examine its cellular localization in vivo. This is particularly important in the case of ubiquitously expressed proteins, such as Id proteins. In this regard, an examination of the in situ localization of Id1 in normal mammary glands reported that Id1 is not expressed in the luminal epithelial cells.
The work defining the role of Id1 in cancers such as breast cancer is limited by the lack of antibodies with suitable sensitivity and specificity to Id1. Sensitivity is an important characteristic for an antibody to detect and/or quantitate Id1, which is very potent and is present in biologic systems at low concentrations [Langlands et al., J Biol Chem; 272: 19785-19793 (1997)]
Production of monoclonal antibodies to the Id1 is difficult because of the relatively low molecular weight (about 17 kDa) and common structural homology among the Id proteins [Nagata and Todokoro, BioChem Biophys Res Commun; 30: 1355-1362 (1994) and Andres-Barquin et al., Histol Histopathol; 15; 603-618 (2000)]. Commercial rabbit polyclonal anti-mouse/human Id1 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.), and commercial mouse monoclonal anti-mouse Id1 antibody (BD PharMingen Corp., San Diego, Calif.) do not have desired binding specificity since it has cross-reactivity with other proteins and does not have high affinity to Id1. The undesired cross-reactivity and low binding affinity make the commercial antibodies unsuitable for use in immunoassays to detect Id1. It is also difficult to prepare polyclonal antibodies specific only to human Id1, with no cross-reactivity to mouse Id1; or specific only to mouse Id1, with no cross-reactivity to human Id1 because of the homology between mouse Id1 and human Id1.
There is a need for antibodies specific for Id1, which do not substantially cross-react with other endogenous proteins or those within the Id family proteins such as Id1, Id2, and Id4. There is also a need for antibodies that have high affinity to Id1 such that they are sensitive to detect or quantitate Id1 in biological samples. There is further a need for antibodies that are specific only to mouse Id1 or specific only to human Id1 with no substantial cross-reactivity between the two species.